| Radix isatidis,the dried root of the cruciferae plant Isatis tinctoria L.,is widely distributed in East Asia and Europe.Among the active ingredients of Radix isatidis,rutin,quercetin,kaempferol and isorhamnetin are polyhydroxyflavonoids with various pharmacological activities,and have been reported to show good preventive and therapeutic potential in the treatment of COVID-19.However,the content of active ingredients in I.tinctoria cultivated in conventional field is prone to fluctuate due to variety,region and environmental factors,which cannot meet the current requirements of the quality of I.tinctoria in the pharmaceutical market.Therefore,we have successfully established an efficient system of I.tinctoria hairy root cultures（I.tinctoria hairy root cultures,ITHRCs）for producing active components of flavonoids secondary metabolism.On this basis,we aimed to use LED light as a mild abiotic inducer to irradiate ITHRCs,and to investigate the effects of red-green-blue mixed light,red,green,blue,and white light on the growth,adventitious bud formation,and accumulation of polyhydroxyflavonoids（rutin,quercetin,kaempferol and isorhamnetin）of ITHRCs.The optimal light culture conditions were optimized and determined,and the changes of physiological and biochemical indexes of ITHRCs under the most appropriate light conditions were measured.The expression changes of key transcription factors involved in the light signaling pathway,flavonoid biosynthetic enzyme genes and transcription factors related to adventitious bud formation in ITHRCs were analyzed by q RT-PCR.The aim of this study was to clarify the potential molecular mechanism of LED light-induced regulation of flavonoid biosynthesis and adventitious bud formation in ITHRCs.The main research content and results of this paper are as follows:1.The effects of different LED light on ITHRCs growth,adventitious bud formation,and polyhydroxyflavonoid accumulation were investigatedCompared with the control（dark culture）,blue light and mixed light significantly promoted the biomass accumulation of ITHRCs.In addition,adventitic bud formation was observed in all ITHRCs grown under LED light.UPLC-MS/MS analysis showed that blue light treatment was more beneficial to the accumulation of target flavonoids in ITHRCs than other light materials.According to the comparison of different light qualities,blue light was the most suitable LED light quality for promoting the growth,adventitious bud formation and flavonoid accumulation of ITHRCs.2.The effects of different initial inoculum sizes on ITHRCs growth,adventitious bud formation,and polyhydroxyflavonoid accumulation under blue light treatment were investigatedWhen ITHRCs with different initial inoculations（0.1%,0.2%,0.3%and 0.4%）were exposed to blue light for 70 days,the bioaccumulation of ITHRCs with higher initial inoculations（0.2%,0.3%and 0.4%）was relatively higher.The initial inoculum of 0.2%ITHRCs was the most conducive to the formation of adventitious shoots under blue light treatment,and the biomass of hairy roots and the yield of flavonoids were at a high level under blue light treatment for 50 days.Compared with the control,the biomass of ITHRCs（7.15±0.63 g/L）was increased by 1.86 folds under the optimal LED light conditions（blue light,initial inoculum amount of 0.2%and treatment time of 50 days）.Rutin（320.49±27.56μg/g DW）,quercetin（388.75±9.17μg/g DW）,kaempferol（787.90±83.43μg/g DW）and isorhamnetin（269.11±20.08μg/g DW）increased by 4.15,9.31,9.09 and 2.88 folds,respectively.3.The changes of physiological and biochemical parameters in ITHRCs cultured under optimal LED light conditions were studiedBy determining the changes in the content of free amino acids（Free amino acids,FAA）and reducing sugars（Reducing sugar,RS）in ITHRCs cultured under optimal light conditions,a clear transition from primary to secondary metabolism occurred in hairy roots at 40 days.The levels of hydrogen peroxide（Hydrogen peroxide,H2O2）and malondialdehyde（Malonic dialdehyde,MDA）were increased in ITHRCs cultured under optimal light condition,which was accompanied by the enhanced activities of catalase（Catalase,CAT）and peroxidase（Peroxidase,POD）.These results indicated that long-term LED blue light exposure could trigger photooxidative stress in ITHRCs.4.The expression levels of key transcription factors of light signaling pathway,flavonoid biosynthetic enzyme genes and transcription factors related to adventitious bud formation in ITHRCs cultured under the optimal LED light condition were investigatedThe results of q RT-PCR analysis showed that the expression levels of cryptochrome gene CRY1,key transcription factors of light signaling pathway HY5,MYB4,and flavonoid biosynthesis related genes PAL,C4H,4CL,CHS,CHI,FLS,and F3’H in ITHRCs were significantly up-regulated from days 30 to 50.Among them,CHS gene（up-regulated 17.58-fold）and FLS gene（up-regulated 13.26-fold）showed higher transcript abundance.Our results showed that LED blue light significantly activated the photoreceptor CRY1 and the key regulator HY5in the light signaling pathway,which resulted in the up-regulation of MYB4 structural genes（such as CHS and FLS）that regulate flavonoid biosynthesis.In addition,the results also indicated that the CUC1 gene（up-regulated 7.09-fold）may be closely related to the blue-light-regulated differentiation and formation of adventitial buds in ITHRCs.In summary,the present study showed that the initial inoculum of 0.2%ITHRCs under LED blue light irradiation for 50 days could significantly increase hairy root biomass production and yield of polyhydroxyflavonoids（rutin,quercetin,kaempferol and isorhamnetin）.Long-term blue light treatment could trigger the photooxidative stress response of ITHRCs and activate the light signal transduction pathway to regulate the expression of flavonoid biosynthesis genes,thereby promoting the accumulation of target flavonoids.In addition,the transcription factors that may be involved in the blue-light-induced adventitious bud formation of ITHRCs were identified. |
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